Francois X. Claret

Trastuzumab: Updated Mechanisms of Action and Resistance in Breast Cancer

HER2-positive breast cancer accounts for 20–30% of all breast cancers and has the second-poorest prognosis among breast cancer subtypes. The approval of trastuzumab in 1998 has significantly improved patients’ outcomes and paved the way for the beginning of advent of targeted approaches in breast cancer treatment. However, primary or acquired resistance to trastuzumab has been increasingly recognized as a major obstacle in the clinical management of this disease. In addition, in clinical practice, there are currently no conclusive biomarkers for patient response to trastuzumab. Therefore, understanding the molecular mechanism of trastuzumab and the development of resistance to this drug are of interest. Such understanding will provide the guidance critically needed for the design of better combination therapy and will allow the appropriate selection of patients who are responsive to trastuzumab-based strategies. In line with that, our review highlights the well-accepted mechanisms of action and resistance to the therapy and discusses the progress that has been made toward successfully overcoming this resistance.

Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase–Positive Anaplastic Large Cell Lymphoma

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) resulting in aberrant expression of chimeric nucleophosmin-ALK. Previously, nucleophosmin-ALK has been shown to activate phosphatidylinositol 3-kinase (PI3K) and its downstream effector, the serine/threonine kinase AKT. In this study, we hypothesized that the mammalian target of rapamycin (mTOR) pathway, which functions downstream of AKT, mediates the oncogenic effects of activated PI3K/AKT in ALK+ ALCL. Here, we provide evidence that mTOR signaling phosphoproteins, including mTOR, eukaryotic initiation factor 4E-binding protein-1, p70S6K, and ribosomal protein S6, are highly phosphorylated in ALK+ ALCL cell lines and tumors. We also show that AKT activation contributes to mTOR phosphorylation, at least in part, as forced expression of constitutively active AKT by myristoylated AKT adenovirus results in increased phosphorylation of mTOR and its downstream effectors. Conversely, inhibition of AKT expression or activity results in decreased mTOR phosphorylation. In addition, pharmacologic inhibition of PI3K/AKT down-regulates the activation of the mTOR signaling pathway. We also show that inhibition of mTOR with rapamycin, as well as silencing mTOR gene product expression using mTOR-specific small interfering RNA, decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis in ALK+ ALCL cells. Cell cycle arrest was associated with modulation of G(1)-S-phase regulators, including the cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Apoptosis following inhibition of mTOR expression or function was associated with down-regulation of antiapoptotic proteins, including c-FLIP, MCL-1, and BCL-2. These findings suggest that the mTOR pathway contributes to nucleophosmin-ALK/PI3K/AKT-mediated tumorigenesis and that inhibition of mTOR represents a potential therapeutic strategy in ALK+ ALCL.

Mechanisms for Lysophosphatidic Acid-induced Cytokine Production in Ovarian Cancer Cells

A potential role for lysophosphatidic acid (LPA) in human oncogenesis was first suggested by the observation that LPA is present at elevated levels in ascites of ovarian cancer patients. In the current study, we demonstrated that LPA is a potent inducer of interleukin-6 (IL-6) and interleukin-8 (IL-8) production in ovarian cancer cells. Both IL-6 and IL-8 have been implicated in ovarian cancer progression. We characterized the IL-8 gene promoter to ascertain the transcriptional mechanism underlying LPA -induced expression of these cytokines. LPA stimulated the transcriptional activity of the IL-8 gene with little effect on IL-8 mRNA stability. The optimal response of the IL-8 gene promoter to LPA relied on binding sites for NF-κB and AP-1, two transcription factors that were strongly activated by LPA in ovarian cancer cell lines. Positive regulators of the NF-κB and AP-1 pathways synergistically activated the IL-8 gene promoter. Further, the effect of LPA on IL-6 and IL-8 generation is mediated by the Edg LPA receptors as enforced expression of LPA receptors restored LPA-induced IL-6 and IL-8 production in non-responsive cells and enhanced the sensitivity to LPA in responsive cell lines. The LPA2 receptor was identified to be the most efficient in linking LPA to IL-6 and IL-8 production although LPA1 and LPA3 were also capable of increasing the response to a certain degree. These studies elucidate the transcriptional mechanism and the Edg LPA receptors involved in LPA-induced IL-6 and IL-8 production and suggest potential strategies to restrain the expression of these cytokines in ovarian cancer.

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition.

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E·CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin®) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.

2-acetylaminofluorene up-regulates rat mdr1b expression through generating reactive oxygen species that activate NF-κB pathway

Overexpression of multidrug resistance genes and their encoded P-glycoproteins is a major mechanism for the development of multidrug resistance in cancer cells. The hepatocarcinogen 2-acetylaminofluorene (2-AAF) efficiently activates rat mdr1b expression. However, the underlying mechanisms are largely unknown. In this study, we demonstrated that a NF-κB site on the mdr1b promoter was required for this induction. Overexpression of antisense p65 and IκBα partially abolished the induction. We then delineated the pathway through which 2-AAF activates NF-κB. 2-AAF treatment led to the increase of intracellular reactive oxygen species (ROS) which causes activation of IKK kinases, degradation of IκBβ (but not IκBα), and increase in NF-κB DNA binding activity. Consistent with the idea that ROS may participate inmdr1b regulation, antioxidant N-acetylcysteine inhibited the induction of mdr1b by 2-AAF. Overproduction of a physiological antioxidant glutathione (GSH) blocked the activation of IKK kinase complex and NF-κB DNA binding. Based on these results, we conclude that 2-AAF up-regulates mdr1b through the generation of ROS, activation of IKK kinase, degradation of IκBβ, and subsequent activation of NF-κB. This is the first report that reveals the specific cis-elements and signaling pathway responsible for the induction of mdr1b by the chemical carcinogen 2-AAF.

JAB1/CSN5: a new player in cell cycle control and cancer

Abstractc-Jun activation domain-binding protein-1 (Jab1) acts as a modulator of intracellular signaling and affects cellular proliferation and apoptosis, through its existence as a monomer or as the fifth component of the constitutive photomorphogenic-9 signalosome (CSN5). Jab1/ CSN5 is involved in transcription factor specificity, deneddylation of NEDD8, and nuclear-to-cytoplasmic shuttling of key molecules. Jab1/CSN5 activities positively and negatively affect a number of pathways, including integrin signaling, cell cycle control, and apoptosis. Also, more recent studies have demonstrated the intriguing roles of Jab1/CSN5 in regulating genomic instability and DNA repair. The effects of Jab1/CSN5s multiple protein interactions are generally oncogenic in nature, and overexpression of Jab1/CSN5 in cancer provides evidence that it is involved in the tumorigenic process. In this review, we highlight our current knowledge of Jab1/CSN5 function and the recent discoveries in dissecting the Jab1 signaling pathway. Further, we also discuss the regulation of Jab1/CSN5 in cancers and its potential as a therapeutic target.

JunB expression is a common feature of CD30+ lymphomas and lymphomatoid papulosis

JunB is a member of the Jun family of proteins that are components of the AP-1 transcription factor complex. AP-1 is involved in cell proliferation and apoptosis. Recent evidence suggests that Hodgkin and Reed–Sternberg cells overexpress JunB and that JunB facilitates constitutive CD30 expression by binding to an AP-1 site in the CD30 promoter. In this study we surveyed JunB expression in a variety of CD30+ lymphoma types including 42 cases of anaplastic large cell lymphoma, 36 classical Hodgkin lymphoma, 15 cutaneous anaplastic large cell lymphoma, and 11 CD30+ diffuse large B-cell lymphoma. In addition, seven cases of nodular lymphocyte-predominant Hodgkin lymphoma and 42 diffuse large B-cell lymphoma, known to be CD30−, were analyzed. JunB expression was assessed using tissue microarrays, immunohistochemistry and a monoclonal antibody specific for JunB. Expression of JunB was observed in 41 of 42 cases of anaplastic large cell lymphoma, including all 21 cases positive for anaplastic lymphoma kinase and 20 of 21 (95%) negative for anaplastic lymphoma kinase. JunB was also expressed in all cases of classical Hodgkin lymphoma, cutaneous anaplastic large cell lymphoma and CD30+ diffuse large B-cell lymphoma, and in lymphomatoid papulosis. By contrast, all nodular lymphocyte-predominant Hodgkin lymphomas and diffuse large B-cell lymphomas that were CD30− were also JunB−. We conclude that JunB is expressed in virtually all CD30+ lymphomas and is a potential target for experimental therapy in patients with these tumors.

Suppression of Jab1/CSN5 induces radio- and chemo-sensitivity in nasopharyngeal carcinoma through changes to the DNA damage and repair pathways.

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus-associated malignancy most common in East Asia and Africa. Radiotherapy and cisplatin-based chemotherapy are the main treatment options. Unfortunately, disease response to concurrent chemoradiotherapy varies among patients with NPC, and many cases are resistant to cisplatin. Increased DNA damage repair is one of the mechanisms contributing to this resistance. Jab1/CSN5 is a multifunctional protein that participates in controlling cell proliferation and the stability of multiple proteins. Jab1 overexpression has been found to correlate with poor prognosis in several tumor types. However, the biological significance of Jab1 activity in response to cancer treatment is unclear. In this study, we used three NPC cell lines (CNE1, CNE2 and HONE1) to investigate the hypothesis that Jab1 positively regulates the DNA repair protein Rad51 and, in turn, cellular response to treatment with DNA-damaging agents such as cisplatin, ionizing radiation (IR) and ultraviolet (UV) radiation. We found that Jab1 was overexpressed in two relatively cisplatin-, IR- and UV-resistant NPC cell lines, and knocking down its expression conferred sensitivity to cisplatin, IR and UV radiation. By contrast, exogenous Jab1 expression enhanced the resistance of NPC cells to cisplatin, IR and UV radiation. Moreover, we provide a mechanism by which Jab1 positively regulated Rad51 through p53-dependent pathway, and increased ectopic expression of Rad51 conferred cellular resistance to cisplatin, IR and UV radiation in Jab1-deficient cells. Taken together, our findings suggest that Jab1 has an important role in the cellular response to cisplatin and irradiation by regulating DNA damage and repair pathways. Therefore, Jab1 is a novel biomarker for predicting the outcome of patients with NPC who are treated with DNA-damaging agents.

Essential Roles of Jab1 in Cell Survival, Spontaneous DNA Damage, and DNA Repair

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to γ-radiation-induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibits its activity and represents a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.

Jab1/CSN5 Negatively Regulates p27 and Plays a Role in the Pathogenesis of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy most common in East Asia and Africa. Aberrant expression of Jab1/CSN5, a negative regulator of the cell-cycle inhibitor p27, is correlated with reduced p27 expression and associated with advanced tumor stage and poor prognosis in several human cancers. In this study, we examined the functional relationship between Jab1 and p27 protein expression in NPC. Immunohistochemical analysis showed an inverse association between Jab1 and p27 in NPC tissue samples, and overexpression of Jab1 correlated with poor survival in patients with NPC. Mechanistically, Jab1 and p27 were found to interact directly in NPC cells, with Jab1 mediating p27 degradation in a proteasome-dependent manner. Knockdown of Jab1 resulted in a remarkable increase in p27 levels and inhibition of cell proliferation, indicating that Jab1 targets p27 for degradation, thereby controlling its stability. Jab1 depletion also enhanced the antitumor effects of cisplatin in NPC cells. Together, our findings suggest that Jab1 overexpression plays an important role in the pathogenesis of NPC through Jab1-mediated p27 degradation. Jab1 therefore represents a novel diagnostic marker and therapeutic target in patients with NPC.